In medical diagnosis, one crucial step influencing the results of a diagnostic method applied is the storage and transportation of the sample. This is especially due to the fact that the analytes that are to be detected within a body sample may be subject to degradation or may otherwise be influenced during the transport and storage. In order to ensure accurate determination of diagnostically relevant analytes the integrity of such analytes within the samples must be maintained until analysis is performed.
In the art, one approach to overcome stability problems for analytes seeks to avoid any transportation and storage of samples by implementing point of care testing systems that allow for analysis of body samples directly at the site of patient care, where the sample has been obtained. However, such approaches are limited respective the analytical technologies that may be applied. For complex analytical methods requiring laboratory equipment, ways to enable transportation and storage of samples without interfering with the integrity of analytes must be found.
One way to allow for transportation of body samples is to preserve the cells contained within the samples and thus to enable for cell based cytological or histological examination of the sample material. Examples for preservation solutions for cytological examinations comprise Digene's Universal Collection Medium (WO9931273), Cytyc®'s PreservCyt® Solution (EP0511430), or Surepath® Cytorich® solution. These media are designed to preserve cellular morphology and also the integrity of cellular proteins to allow for cytological examination of the preserved cellular samples. All such preservation solutions comprise alcohols as fixatives. Generally spoken, preservation of the analytes in such procedures is achieved by addition of chemical preservative substances. In cases where the cellular integrity is no longer needed for the diagnostic procedure the integrity of the analytes in a solution may similarly be achieved by addition of chemical preservative substances. The main disadvantage of this approach is that preservatives are often toxic substances that may harm operators and may damage the environment in case of spill. Additionally the requirements for waste disposal as well as the cost for waste disposal are increased when using chemical additives for sample stabilization.
One further approach to solve the problem of stability of analytes in body samples is refrigeration or freezing of the body samples during transportation and storage. It is known that biological substances may be preserved by refrigeration for a certain period of time and that freezing of biological material below −20° C. may be applied for preservation of long term storage. This solution however has the disadvantage of energy consumption and posing hurdles on the transportation process. It cannot always be ensured that refrigeration or maintaining temperatures below −20° C. may be upheld throughout the transportation. This turns out to be a serious problem in cases where the stability of an analyte is not proven under certain temperature conditions. Therefore the method of the present invention involving a heating step provides an easy way to stabilize samples for transportation and storage without the need for addition of preservatives or the need for refrigeration.
Additionally, the heating step if performed in a medium that comprises denaturing agents may contribute to the reproducibility and accuracy of subsequent analytical steps. If the heating step is e.g. performed in the presence of Sodium Dodecyl Sulfate that interacts with proteins in the respective sample, a denatured structure of the proteins is obtained. This denatured structure may in certain cases be of advantage for the reproducible and quantitative determination of the protein in solution.
In the art, methods for denaturing protein bio-assays are used in the case of the Laemmli-System in denaturing Polyacrylamide Gel Electrophoresis in the presence of SDS (known as SDS PAGE). In this case the samples containing proteins are also heated in the presence of SDS to allow denaturation of the protein and are subsequently separated according to their size in an electrophoretic step. The purpose of the denaturation in this method is denaturation of the protein to allow for an overall linearization of the proteins together with homogeneous loading of the proteins with SDS ions. This ensures that proteins in contrast to their native form exhibit an electrical charge that is proportional to the overall size of the molecules. The overall method allows for separation of the molecules by size.
The present invention is based on the inventor's findings that analytes in solubilzed body samples may be stabilized by heating of the sample solution for a certain period of time. According to the present invention heating may for example be performed in a suitable sample medium. The method according to the present invention overcomes the drawbacks of the methods known in the art. The method found by the inventors does not make use of chemical preservatives that may cause harm to animals, human beings or the environment and does not impose the need for refrigeration or keeping up of other energy consuming conditions during the whole process of transportation and storage.
The effect of denaturing of the proteins during the heating step allows for accurate determination of the proteins within the samples using denaturing bioassays. In contrast to the methods known in the art, the denaturing immunoassay is immunochemical detection directly from the sample solution employing a solid phase fixed detection probe that specifically binds to the proteins within the sample. An electrophoretic step is not necessarily comprised in the overall method. The purpose and effect of the denaturation in the context of this method is therefore not due to the generation of homogeneous size dependant electric charge of the proteins but is in part due to the linearization and therefore to improved accessibility of antigenic epitopes within the proteins.